Endothelial Progenitor Cells Affect the Growth and Apoptosis of Renal Cells by Secreting Microvesicles Carrying Dysregulated miR-205 and miR-206

Background This study investigated the mechanism of microRNA (miRNA, miR) in microvesicles (MVs) secreted by endothelial progenitor cells (EPCs) involved in renal function in vivo and in vitro injury repair of rat primary kidney cells (PRKs). Methods Gene Expression Omnibus analysis of potential target miRNAs in nephrotic rats. Real-time quantitative polymerase chain reaction verified the correlation of these miRNAs and screened the effective target miRNAs and their downstream putative target mRNAs. Western blot analyzes the protein levels of DEAD-box helicase 5 (DDX5) and the activation of the proapoptotic factor caspase-3/9 (cleaved). Dil-Ac-LDL staining, immunofluorescence, and a transmission electron microscope (TEM) were used to identify the successful isolation of EPCs and PRKs and the morphology of MVs. Cell Counting Kit-8 was used to detect the effect of miRNA-mRNA on the proliferation of PRKs. Standard biochemical kits were used to detect biochemical indicators in rat blood and urine. Dual-luciferase analysis of miRNA binding to mRNA was conducted. The effect of miRNA-mRNA interaction on the apoptosis level of PRKs was analyzed by flow cytometry. Results A total of 13 rat-derived miRNAs were potential therapeutic targets, and miR-205 and miR-206 were screened as the targets of this study. We found that the EPC-MVs alleviated the increase of blood urea nitrogen and urinary albumin excretion and the decrease in creatinine clearance caused by hypertensive nephropathy in vivo. The effect of MVs in improving renal function indicators was promoted by miR-205 and miR-206 and inhibited by knockdown of expressed miR-205 and miR-206. In vitro, angiotensin II (Ang II) promoted growth inhibition and apoptosis of PRKs, and similarly, dysregulated miR-205 and miR-206 affected the induction of Ang II. We then observed that miR-205 and miR-206 cotargeted the downstream target DDX5 and regulated its transcriptional activity and translational levels, while also reducing the activation of proapoptotic factors caspase-3/9. Overexpressed DDX5 reversed the effects of miR-205 and miR-206. Conclusion By upregulating the expression of miR-205 and miR-206 in MVs secreted by EPC, the transcriptional activity of DDX5 and the activation of caspase-3/9 can be inhibited, thereby promoting the growth of PRKs and protecting the injury caused by hypertensive nephropathy.


Introduction
The kidney is one of the major organs affected by hypertensive target organ damage [1,2]. Hypertensive nephropathy (HN) is clinically characterized by progressive renal fibrosis and inflammation [3,4], and prolonged hypertension can eventually lead to renal failure [4]. However, the current treatment of HN is still at the level of eliminating symptoms, and the research on the pathogenesis of HN is still unclear. It is generally believed that urinary albumin excretion ðUAEÞ > 20 mg/24 h in Spontaneous Hypertension Rat (SHR) can be considered HN.
Endothelial progenitor cells (EPCs) are a type of stem cells with angiogenic and tissue repair capabilities [5].
Evidence shows that endothelial progenitor cells can improve renal function in patients with diabetic nephropathy [6], possibly because of the beneficial therapeutic effects of endothelial progenitor cell-derived microvesicles (MVs) in various diseases [7]. Many reports have confirmed that the protective effect of EPCs is closely related to the release of MVs [8]. The MVs secreted by EPCs can be absorbed by cells, thereby reducing damage [9] and repairing tissues [10]. The properties of EPC-MVs are similar to those of EPCs. However, the underlying molecular mechanism of EPC repairing renal injury through MVs is still unclear.
MicroRNAs (miRNA, miR) are small noncoding RNAs involved in the progression and treatment of various diseases including HN [11,12]. Evidence suggests that MVs can ameliorate renal injury by delivering miRNAs [13,14]. It is well known that dysregulated miRNAs can affect the occurrence and development of various diseases [15,16], including HN [17]. miRNAs affect renal function by binding to the 3 ′ -UTR of downstream mRNAs and regulating mRNA transcription and translation [18,19]. The levels of angiotensin II (Ang II) and its receptors in the kidneys of hypertensive rats are much higher than those of Wistar-Kyoto (WKY) rats [20]. Ding et al. believed that angiotensin II-mediated urinary albumin, blood urea nitrogen (BUN), and creatinine clearance (Scr) can be improved by miR-101a by blocking nuclear factor kappa-B signaling [3]. Downregulated miR-205 was positively correlated with BUN and creatinine (Cr) levels in patients with renal injury [21]. After overexpression, miR-205 attenuated sepsis-induced acute kidney injury [22]. miR-206 binds to DEAD-box helicase 5 (DDX5) to inhibit the activation of NLR family pyrin domain containing 3 inflammasome and alleviate acute kidney injury [23].
These proteases, cysteinyl aspartate-specific proteinase (caspase) family of cysteine proteases, are key enzymes that cause apoptosis; once the caspase is activated, a cascade of caspase ensues, eventually leading to apoptosis. Evidence shows that, as important members of the caspase family, the activation of caspase-3 and caspase-9 is the key to affecting renal cell apoptosis [24,25] and promotes the extent of damage to kidney cells in response to Ang II, chemotherapy drugs, or oxidative stress [26][27][28].
Studies have shown that renal injury caused by hypertension is related to the activation of the apoptosis pathway (caspase-3/9) [29]. Kidney tissue damage can be significantly alleviated and prevented by reducing the activity of apoptotic proteins such as cleaved caspase-9 and cleaved caspase-3 [30]. Proteins that promote apoptosis can exacerbate kidney damage caused by inflammation, oxidative stress, or high blood sugar and hypertension [31][32][33]. This study attempts to explore the mechanism by which miR-205 and miR-206 protect the kidneys from damage caused by hypertension from the perspective that EPC-MVs can carry dysregulated miRNAs to affect disease.

2.
1. Animals. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Six Wistar-Kyoto-and SHR-(male, 10-week-old) specific pathogen-free rats weighing 300-330 g were obtained from the Beijing Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). Wistar-Kyoto rats were used as the control group, and SHR rats as the model group. All rats were placed in a room with 12 h light/dark cycle, the temperature was maintained at 25°C, and the humidity was maintained at 55%; rats have ad libitum access to standard rat food and water in a polystyrene cage. At the end of the experiment, the rats were sacrificed by intraperitoneal injection of sodium pentobarbital (200 mg/kg body weight). Animal experiments were approved by the Animal Care and Use Committee of Hainan Medical University (Haikou, China, approval no. HYLL-2021-053) and were conducted according to the National Institutes of Health guidelines.

Biochemical
Analysis of Blood and Urine. On the day before blood sample collection, urine samples were continuously collected from each animal for 24 hours. Blood urea nitrogen (BUN), serum creatinine (Scr), and UAE were measured by standard biochemical kits (BHKT Clinical Reagent Co., Ltd., Beijing, China). Creatinine clearance (CCr) was calculated according to the following formula: CCr = urinary creatinine ðmg/mlÞ × urine output ðml/kgÞ/ plasma creatinine ðmg/mlÞ [34].
2.4. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). According to the manufacturer's instructions, PRKs, EPCs, and MVs were extracted using Trizol reagent (Invitrogen, Thermo Fisher Scientific, Inc.). After 10 minutes of centrifugation (13,000 × g, 4°C) using JIDI-17RS refrigerated centrifuge (Guangzhou JIDI Instrument Co., Ltd, Guangzhou, China), RNA was reverse-transcribed with PrimeScript RT kit (Takara Bio, Japan). SYBR® Premix Ex TaqTM II kit (Takara Bio) was used for RT-qPCR analysis using the Applied Biosystems®7500 Real-Time PCR system (CA, USA). PCR experiments were carried out under the following conditions: 50°C for 25 min, 94°C for 2 min, followed by 25 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 2 min. The primer sequences of miR-205, miR-206, and DDX5 used for RT-qPCR are shown in Table 1. Target RNA levels were normalized to those of the housekeeping genes U6 or β-actin, and relative levels of miR-205, miR-206, and DDX5 expression were determined using the 2 −ΔΔCt method [35].
2.5. Culture and Identification of EPCs. After dissociation of rat femur and tibia, the mixture was rinsed with sterile PBS and collected. Following centrifugation at 4°C (1000 × g,

Cell Proliferation Assays.
According to the manufacturer's instructions, 10 μl of Cell Counting Kit-8 reagent (CCK-8, Solarbio) was added to PRKs at 0 h and 24 h, respectively, and incubated for 1 h. The optical density at 490 nm was then measured with a microplate reader (Multiskan MK3, Thermo Fisher Scientific, Inc.) to assess the cell proliferation rate.

Dual
Luciferase. The miRDB database (http://mirdb .org/) was used to predict binding sites between miR-205 or miR-206 and DDX5. PRKs were transfected with miR-205 or miR-206 and their negative controls, vector plasmid containing wild-type (WT) or mutant (mut) DDX5, and pRL-SV40 reporter vector plasmid. Transfected cells were incubated for 48 h, and luciferase activity was measured at 490 nm according to dual-luciferase reporter assay system (Promega, USA) instructions.
2.14. Statistical Analyses. All experiments were repeated three times. Data are expressed as the mean ± standard deviation (SD). Differences between multiple groups were assessed using one-way analysis of variance and Bonferroni post hoc test. Student's t-test was used for independent two-group analyses. Statistical significance was set at p < 0:05.

EPC Isolation and
Identification. Isolated EPCs were identified using Dil-Ac-LDL staining. The red fluorescence (Dil-Ac-LDL) coincided with blue fluorescence (DAPI nuclear staining) by more than 90% (Figure 2(a)), confirming the successful separation of EPCs. After the medium was collected, MVs were extracted and observed under TEM. TEM results observed double-membrane vesicle-like bodies (Figure 2(b)). After western blot analysis, the protein levels of CD63, CD81, and Tsg101 were all positive (Figure 2(c)), so we determined that MVs were successfully extracted. miR-205 and miR-206 construct agomiR/antago-miR transfected EPC and collected supernatant and secreted MVs. The results showed that miR-205 and miR-206 were upregulated in the agomiR-transfected group and downregulated in the antagomiR-transfected group regardless of EPCs, supernatant, or MVs (Figures 2(d)-2(i)). Therefore, we confirmed that the synthetic miR-205 and miR-206 agomiR/

Disease Markers
PRKs, indicating that the isolation of PRKs was successful (Figure 4(a)). When MVs labeled with PKH26 were added to PRKs, we observed that red fluorescence of PKH26 was detected in the cytoplasm of PRKs, confirming that MVs could be incorporated into PRKs (Figure 4  3.7. Effects of the miR-205/miR-206-DDX5 Axis on Proapoptotic Proteins. By western blot analysis, we confirmed that Ang II promoted DDX5 protein level and the activation of proapoptotic protein caspase-3/9, and EPC-MVs had a significant inhibitory effect on this effect. The efficacy of EPC-MVs was enhanced in the overexpressed state of miR-205 or miR-206 and was attenuated by overexpression of DDX5 ( Figure 6). Furthermore, EPC-MVs inhibited Ang II-mediated PRK growth inhibition and apoptosis promotion. miR-205 or miR-206 enhanced the utility of EPC-MVs, whereas overexpression of DDX5 inhibited the utility of miR-205 or miR-206 MVs (Figures 7(a)-7(d)).

Discussion
In the present study, we demonstrated for the first time that EPC-MVs carrying high levels of miR-205 and miR-206 could protect kidneys or PRKs from cellular damage caused by hypertension or Ang II. The proliferation and angiogenesis and anti-inflammatory abilities of EPCs can effectively protect the kidneys of patients with nephropathy [37,38] and play an important role in maintaining vascular integrity and improving renal function [6]. Recent evidence suggests that EPCs play a role in protecting renal function through secreted MVs [7]. MVs are antiinflammatory, improve endothelial function, and alleviate endothelial dysfunction caused by oxidative stress [39,40]. Since the damaged kidneys cannot effectively filter the metabolic wastes in the blood, which eventually leads to the occurrence of kidney disease [41], this paper conducted a GEO analysis on the diabetic rats based on the possibility of kidney disease in the diabetic rats, and the data was used to analyze the same kidney damage caused by hypertension.
One of the main conclusions of this study is that MVs secreted by EPCs reduced the rise in UAE and BUN and the decrease in CCr induced by HN in vivo. In vitro, MVs promoted the proliferation of PRKs and inhibited apoptosis; all these evidences confirmed that MVs can improve renal function. This is consistent with the conclusions reached by our previous study [37].
It has been reported that MVs can deliver miRNAs to cells, thereby affecting disease development. For example, MVs carrying miR-148a can regulate adipogenic and osteogenic differentiation by targeting the receptor tyrosine kinase-like orphan receptor 2 pathway [42]. miR-191 secreted by MVs induces renal tubular epithelial cell apoptosis by inhibiting cystathionine-β-synthase [43]. This study confirmed that both miR-205 and miR-206 secreted by MVs could promote the improvement of HN by MVs. The secreted low-expressed miR-205 and miR-206 inhibited the effect of MVs. The results in in vitro experiments were also similar; to be specific, MVs secreted miR-205 and miR-206 to promote MVs to ameliorate Ang II-induced PRK growth

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Disease Markers inhibition and apoptosis, and low expression of miR-205 and miR-206 partially inhibited the effect of MVs. Furthermore, after transfection of miR-205 or miR-206 agomiR/ antagomiR, deregulated expression of miR-205 or miR-206 was detected in EPCs, supernatants, and MVs, implying that MVs carried deregulated miR-205 or antagomiR miR-206 is produced by secretion from EPCs.
To further explore the mechanism by which miR-205 or miR-206 exert their effects, we analyzed their common downstream target DDX5 by bioinformatics. Studies have confirmed that miR-206 improves renal function by binding to DDX5 [23]. Here, we report that miR-205 and miR-206 together target DDX5. The growth promotion and inhibition of PRKs by miR-205 and miR-206 could be reversed by overexpressed DDX5, and the restriction of transcriptional activity and translational level of DDX5 by miR-205 and miR-206 was also alleviated by overexpressed DDX5. Therefore, DDX5 is negatively regulated with miR-205 and miR-206.
Increasing evidence suggests that abnormal activation of apoptotic proteins may be a major factor in causing kidney damage [31,44]. Our study shows that MVs can reduce the activation of apoptotic protein caspase-3/9. Secretion of miR-205 and miR-206 enhanced the effect of MVs, while overexpression of DDX5 inhibited the enhancement of MVs by miR-205 and miR-206.
This study has some limitations. First, there are no clinical data on the treatment of EPC-MVs in HN. Second, changes in various signaling pathways, especially those related to inflammation or oxidative stress, have not been further explored. All of these will be the focus of our future research.
In conclusion, EPCs protect against hypertensioninduced renal injury by secreting MVs carrying dysregulated miR-205 and miR-206 expression. The mechanism is related to miR-205 and miR-206 cotargeting DDX5 and promoting cell growth inhibition and apoptosis. This provides a new therapeutic strategy for HN.

Data Availability
The data used to support the findings of this study are included within the article.

Ethical Approval
The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Animal Care and Use Committee of Hainan Medical University (Haikou, China, approval no. HYLL-2021-053).

Conflicts of Interest
The author(s) declare(s) that they have no conflicts of interest.